- Email: [email protected]
326 Potentiation of basophil function by antibodies of ICAM-3
264
325
Abstracts
Integrin-dependent histamine release in human b a s o p h i l s : R e l a t i o n s h i p with disease s t a t u s . K
J ALLERGY CLIN IMMUNOL JANUARY 1996
327
MD. DW MacGlashan MD PhD, Baltimore. MD. We have previously shown that stimulation of HB causes changes in total cellular PKC activity. However, there is a family of at least 12 PKC isozymes and the profile of PKC isozymc expression in HB is unknown. Therefore, we have examined HB for the presence of 7 PKC isozymes. HB were purified by a combination of elutriation, discontinuous Percoll density gradients and anti-lgE/MACS bead positive selection: HB purity ranged from 97-99.7%. For comparative purposes, the contaminating cells (CC) from the last step of this purification scheme (a mixture of lymphocytes and monocytes) were also studied for PKC isozyme expression. PKC isozymes were identified by Western blotting proteins obtained from lysed HB or CC. In the context of PKC expression in the CC, the results from HB were interesting. Both HB and CC cells expressed essentially equivalent levels (on a per cell and per/.tg protein basis) of 1~I, ~1I, and ¢ PKC. PKC ~5was expressed at significantly higher levels in HB than in CC. Surprisingly, PKC ct was difficult to detect in HB. A semi-quantitative analysis indicated that PKC ¢t is expressed at ~10% the level found in CC. PKC rl was detectable in CC but barely detectable in HB (_<5% of contaminating cell expression). PKC y was undetectable in both populations of cells. The relative absence of PKC ~ in HB (and the near absence of PKC rl) is interesting for its functional implications. For example, PMA-treatment of HB inhibits ionomycininduced IL-4 secretion while it enhances IL-4 release from ionomycin-stimulatcd lymphocytes. Whether this observation will be the consequence of differential expression of PKC isozymes between HB and lymphocytes remains to be seen but will be the subject of further investigation.
Goldring. K Hurst, R D iukanovic and JA Warner. Southampton, UK. We have investigated the effects of clustering the VLA-4 integdns CD29 and CD49d using either mAb or tissue fibronectin (tFN) on histamine release (HR) from humatl basophils. The non-atopic, non-asthmatic controls (n=8) failed to release his~nine following clustering of CD29 (HR=2+2%), CD49d ( H R = I + I % ) or challenge with 10~g/ml tFN (HR=0+0%), but did respond to anti-IgE (HR=35+7%). In contrast, basophils from asthmatic patients (n=33) responded to CD29(HR=8+2%), CD49d(HR=8+2%) and tFN (HR=7+2%) in addition to anti-lgE (HR=34+5%). Dividing the group into patients with mild or moderate asthma failed to reveal any distinctions between the two groups. When we tested basophils from non-atopic asthmatics (n=8), we found that they failed to respond to either CD29 ( H R = I + I % ) , CD49d (HR=I+0%) or tFN (HR=0+0%). However, these donors did respond to anti-lgE (HR=38+7%). Preliminary studies suggest that patients with severe atopy show some response to integfins, suggesting that integrin dependent release is related to the presence of allergic disease rather than asthmatic status.
326
P o t e n t i a t i o n of Basophil Function by A n t i b o d i e s to I C A M - 3 . S Saini MD. J White MAS. WM Gallatin PhD*, PA H0ffman PhD*. LM Lichtensi¢in MD PhD, BS .B0~;hner MD, Baltimore, MD and *Bothell, WA ICAM-3 is an Ig superfamily ligand for [32 intcgrins. It is a signaling molecule on T cells, enhancing binding to endothelium and matrix proteins, and stimulating proliferation. ICAM-3 is expressed on all leukocytes, but little is known about its function on other non-T cells. We examined the effect of a mAb directed against domain l of ICAM-3 (ICR-2) on human peripheral blood basophil mediator release and adhesion in vitro. Basophils were obtained by various techniques (purity <1 to 66%). Cells were preincubated with or without 1CR-2 and anti-lgEinduced (I-100 ng/ml, 45 rain) histamine release (HR) was determined. ICR-2 (0.2-2 /.tg/ml) potentiated anti-lgEinduced HR (e.g., HR using 0.1 /~g/ml anti-IgE_with or without ICR-2 (2 I~g/ml) was 42+7 and 33=1:5%, X+SEM, respectively, n=10, p<0.02); 1CR-2 alone did not induce HR. Maximal increases in HR occurred within 1 rain and were less or absent with 30 rain of preificubation. ICR-2 augmented HR in both allergic and non-allergic donors regardless of basophil purity. In contrast, ICR-2 did not consistently alter anti-IgE-induced release of LTC4 (45 min) or IL-4 (4 hr). Finally, coincubation with ICR-2 enhanced basophil adherence (10 rain) to both unstimulated and IL* ll3-treated (5 ng/ml, 4 h0 endothelial cells (from 14_+.2 to 22~.5 and from 39-1-6 to 47+1% adhesion, respectively, n=2). Thus, crosslinking of ICAM-3 on basophils augments adhesion and IgE-dependent HR, but not release of other mediators. Engagement of basophil ICAM-3 by 132 integrins on other cells may potentiate basophil function.
Expression of Calcium-Dependent and Independent PKC Isozymes in H u m a n Basophils (HB). K Miura
328
FeeR1 I}-SUBUNIT E X P R E S S I O N IS E N H A N C E D BY IN V I V O A D M I N I S T R A T I O N O F IgE IN R A T S . JS M~rshall. J Rivera, B Hewlett. R Stead. N Shaikh, Hamilton, Ontario, Canada; NIAMS, NIH, Bethesda, MD, USA. Activation of mast cells can occur through cross-linking of FeeRI containing single ~- and fl- and two -Y subunits. We have employed a combination of immunohistochemistry and Alcian blue staining to examine lgE receptor expression by mast cells in rats. B-subonit and IgE expression were detected using monoclonal antibodies JRK and MARE-! respectively and an immunoperoxidase technique. Initial experiments demonstrated that infection with the Nippostrongylus brasiliensis induced an increase in 8-subunit expression by both tongue and jejunal mast cells which was maximal at 14 days post infection. The integrated optical densities GOD) values for fl-subunit staining assessed by image analysis increased from a mean value of 2.0 __+ 1 on day 7 to 48 __+ 12 on day 14 post infection (nffi4). The serum levels of IgE were highly correlated with the amount of B-subunit staining (p < 0.0001). In view of this data, we administered rat myeloma IgE IR162 to animals for 3 days and determined their expression of IgE receptor. In 8 out of 9 IgE injected rats there was a dramatic rise in FceRI Ii-subunit expression (p< 0.01) and an increase in IgE bound to mast ceils. The amount of fI-subunit and IgE staining of mast cells observed was again significantly correlated with serum IgE levels (p < 0.01). These observations suggest that FceRl expression by mast cell may be enhanced in vivo by IgE.
325
Abstracts
Integrin-dependent histamine release in human b a s o p h i l s : R e l a t i o n s h i p with disease s t a t u s . K
J ALLERGY CLIN IMMUNOL JANUARY 1996
327
MD. DW MacGlashan MD PhD, Baltimore. MD. We have previously shown that stimulation of HB causes changes in total cellular PKC activity. However, there is a family of at least 12 PKC isozymes and the profile of PKC isozymc expression in HB is unknown. Therefore, we have examined HB for the presence of 7 PKC isozymes. HB were purified by a combination of elutriation, discontinuous Percoll density gradients and anti-lgE/MACS bead positive selection: HB purity ranged from 97-99.7%. For comparative purposes, the contaminating cells (CC) from the last step of this purification scheme (a mixture of lymphocytes and monocytes) were also studied for PKC isozyme expression. PKC isozymes were identified by Western blotting proteins obtained from lysed HB or CC. In the context of PKC expression in the CC, the results from HB were interesting. Both HB and CC cells expressed essentially equivalent levels (on a per cell and per/.tg protein basis) of 1~I, ~1I, and ¢ PKC. PKC ~5was expressed at significantly higher levels in HB than in CC. Surprisingly, PKC ct was difficult to detect in HB. A semi-quantitative analysis indicated that PKC ¢t is expressed at ~10% the level found in CC. PKC rl was detectable in CC but barely detectable in HB (_<5% of contaminating cell expression). PKC y was undetectable in both populations of cells. The relative absence of PKC ~ in HB (and the near absence of PKC rl) is interesting for its functional implications. For example, PMA-treatment of HB inhibits ionomycininduced IL-4 secretion while it enhances IL-4 release from ionomycin-stimulatcd lymphocytes. Whether this observation will be the consequence of differential expression of PKC isozymes between HB and lymphocytes remains to be seen but will be the subject of further investigation.
Goldring. K Hurst, R D iukanovic and JA Warner. Southampton, UK. We have investigated the effects of clustering the VLA-4 integdns CD29 and CD49d using either mAb or tissue fibronectin (tFN) on histamine release (HR) from humatl basophils. The non-atopic, non-asthmatic controls (n=8) failed to release his~nine following clustering of CD29 (HR=2+2%), CD49d ( H R = I + I % ) or challenge with 10~g/ml tFN (HR=0+0%), but did respond to anti-IgE (HR=35+7%). In contrast, basophils from asthmatic patients (n=33) responded to CD29(HR=8+2%), CD49d(HR=8+2%) and tFN (HR=7+2%) in addition to anti-lgE (HR=34+5%). Dividing the group into patients with mild or moderate asthma failed to reveal any distinctions between the two groups. When we tested basophils from non-atopic asthmatics (n=8), we found that they failed to respond to either CD29 ( H R = I + I % ) , CD49d (HR=I+0%) or tFN (HR=0+0%). However, these donors did respond to anti-lgE (HR=38+7%). Preliminary studies suggest that patients with severe atopy show some response to integfins, suggesting that integrin dependent release is related to the presence of allergic disease rather than asthmatic status.
326
P o t e n t i a t i o n of Basophil Function by A n t i b o d i e s to I C A M - 3 . S Saini MD. J White MAS. WM Gallatin PhD*, PA H0ffman PhD*. LM Lichtensi¢in MD PhD, BS .B0~;hner MD, Baltimore, MD and *Bothell, WA ICAM-3 is an Ig superfamily ligand for [32 intcgrins. It is a signaling molecule on T cells, enhancing binding to endothelium and matrix proteins, and stimulating proliferation. ICAM-3 is expressed on all leukocytes, but little is known about its function on other non-T cells. We examined the effect of a mAb directed against domain l of ICAM-3 (ICR-2) on human peripheral blood basophil mediator release and adhesion in vitro. Basophils were obtained by various techniques (purity <1 to 66%). Cells were preincubated with or without 1CR-2 and anti-lgEinduced (I-100 ng/ml, 45 rain) histamine release (HR) was determined. ICR-2 (0.2-2 /.tg/ml) potentiated anti-lgEinduced HR (e.g., HR using 0.1 /~g/ml anti-IgE_with or without ICR-2 (2 I~g/ml) was 42+7 and 33=1:5%, X+SEM, respectively, n=10, p<0.02); 1CR-2 alone did not induce HR. Maximal increases in HR occurred within 1 rain and were less or absent with 30 rain of preificubation. ICR-2 augmented HR in both allergic and non-allergic donors regardless of basophil purity. In contrast, ICR-2 did not consistently alter anti-IgE-induced release of LTC4 (45 min) or IL-4 (4 hr). Finally, coincubation with ICR-2 enhanced basophil adherence (10 rain) to both unstimulated and IL* ll3-treated (5 ng/ml, 4 h0 endothelial cells (from 14_+.2 to 22~.5 and from 39-1-6 to 47+1% adhesion, respectively, n=2). Thus, crosslinking of ICAM-3 on basophils augments adhesion and IgE-dependent HR, but not release of other mediators. Engagement of basophil ICAM-3 by 132 integrins on other cells may potentiate basophil function.
Expression of Calcium-Dependent and Independent PKC Isozymes in H u m a n Basophils (HB). K Miura
328
FeeR1 I}-SUBUNIT E X P R E S S I O N IS E N H A N C E D BY IN V I V O A D M I N I S T R A T I O N O F IgE IN R A T S . JS M~rshall. J Rivera, B Hewlett. R Stead. N Shaikh, Hamilton, Ontario, Canada; NIAMS, NIH, Bethesda, MD, USA. Activation of mast cells can occur through cross-linking of FeeRI containing single ~- and fl- and two -Y subunits. We have employed a combination of immunohistochemistry and Alcian blue staining to examine lgE receptor expression by mast cells in rats. B-subonit and IgE expression were detected using monoclonal antibodies JRK and MARE-! respectively and an immunoperoxidase technique. Initial experiments demonstrated that infection with the Nippostrongylus brasiliensis induced an increase in 8-subunit expression by both tongue and jejunal mast cells which was maximal at 14 days post infection. The integrated optical densities GOD) values for fl-subunit staining assessed by image analysis increased from a mean value of 2.0 __+ 1 on day 7 to 48 __+ 12 on day 14 post infection (nffi4). The serum levels of IgE were highly correlated with the amount of B-subunit staining (p < 0.0001). In view of this data, we administered rat myeloma IgE IR162 to animals for 3 days and determined their expression of IgE receptor. In 8 out of 9 IgE injected rats there was a dramatic rise in FceRI Ii-subunit expression (p< 0.01) and an increase in IgE bound to mast ceils. The amount of fI-subunit and IgE staining of mast cells observed was again significantly correlated with serum IgE levels (p < 0.01). These observations suggest that FceRl expression by mast cell may be enhanced in vivo by IgE.